The overall objective of this research project is to determine the role in catalysis of the amino acid residues in ribonuclease A (RNase A), and to create mutant and hybrid RNase A's of modified substrate specificity. RNase A is a protein of 124 amino acid residues that efficiently catalyzes the hydrolysis of RNA. The enzyme has a well-defined enzymology and three- dimensional structure, and has been widely used in protein folding studies. As one of the best characterized of all enzymes, RNase A is an ideal object for studying relationships between protein structure and enzyme function. This research project enlists protein engineering to elucidate catalysis by RNase A. The cDNA for RNase A has been cloned and expressed in S. cerevisiae. The specific aims of this research project use mutant RNase A's: (1) to delineate the ability of Lys41 to stabilize the transition states of the catalytic reaction, (2) to determine whether Asp121 of the putative catalytic triad abstracts a proton from His119, or simply serves to stabilize the tautomer of His119 needed for catalysis, (3) to achieve altered substrate specificity (using random mutagenesis followed by phenotypic selection), and (4) to create a hybrid enzyme that catalyzes the hydrolysis of RNA only at the sequence dictated by an attached DNA oligonucleotide. To interpret chemically the measurements of enzyme action, the three-dimensional structure of RNase A mutants will be determined by X-ray diffraction analysis. The proposed experiments use techniques from molecular biology, biochemistry, and biophysical chemistry to test ideas on the relationship between protein structure and enzyme function. The results of these experiments may lead to insights into this relationship, as well as to the creation of novel enzymes for medicinal analyses and therapies, and for industrial processes.